Spinach hybrid RX 06681616 and parents thereof

ABSTRACT

The invention provides seed and plants of spinach hybrid RX 06681616 and the parent lines thereof. The invention thus relates to the plants, seeds and tissue cultures of spinach hybrid RX 06681616 and the parent lines thereof, and to methods for producing a spinach plant produced by crossing such plants with themselves or with another spinach plant, such as a plant of another genotype. The invention further relates to seeds and plants produced by such crossing. The invention further relates to parts of such plants, including the leaf and gametes of such plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. Provisional Appl. Ser. No.61/572,294, filed Aug. 26, 2011, the entire disclosure of which isincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the field of plant breeding and, morespecifically, to the development of spinach hybrid RX 06681616 and theinbred spinach lines SSB66-1090 F and SSB66-1131 M.

BACKGROUND OF THE INVENTION

The goal of vegetable breeding is to combine various desirable traits ina single variety/hybrid. Such desirable traits may include any traitdeemed beneficial by a grower and/or consumer, including greater yield,resistance to insects or pests, tolerance to environmental stress,better agronomic quality, higher nutritional value, growth rate andfruit properties.

Breeding techniques take advantage of a plant's method of pollination.There are two general methods of pollination: a plant self-pollinates ifpollen from one flower is transferred to the same or another flower ofthe same plant or plant variety. A plant cross-pollinates if pollencomes to it from a flower of a different plant variety.

Plants that have been self-pollinated and selected for type over manygenerations become homozygous at almost all gene loci and produce auniform population of true breeding progeny, a homozygous plant. A crossbetween two such homozygous plants of different genotypes produces auniform population of hybrid plants that are heterozygous for many geneloci. Conversely, a cross of two plants each heterozygous at a number ofloci produces a population of hybrid plants that differ genetically andare not uniform. The resulting non-uniformity makes performanceunpredictable.

The development of uniform varieties requires the development ofhomozygous inbred plants, the crossing of these inbred plants, and theevaluation of the crosses. Pedigree breeding and recurrent selection areexamples of breeding methods that have been used to develop inbredplants from breeding populations. Those breeding methods combine thegenetic backgrounds from two or more plants or various other broad-basedsources into breeding pools from which new lines and hybrids derivedtherefrom are developed by selfing and selection of desired phenotypes.The new lines and hybrids are evaluated to determine which of those havecommercial potential.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a spinach plant of thehybrid designated RX 06681616, the spinach line SSB66-1090 F or spinachline SSB66-1131 M. Also provided are spinach plants having all thephysiological and morphological characteristics of such a plant. Partsof these spinach plants are also provided, for example, includingpollen, an ovule, scion, a rootstock, a fruit, and a cell of the plant.

In another aspect of the invention, a plant of spinach hybrid RX06681616 and/or spinach lines SSB66-1090 F and SSB66-1131 M comprisingan added heritable trait is provided. The heritable trait may comprise agenetic locus that is, for example, a dominant or recessive allele. Inone embodiment of the invention, a plant of spinach hybrid RX 06681616and/or spinach lines SSB66-1090 F and SSB66-1131 M is defined ascomprising a single locus conversion. In specific embodiments of theinvention, an added genetic locus confers one or more traits such as,for example, herbicide tolerance, insect resistance, disease resistance,and modified carbohydrate metabolism. In further embodiments, the traitmay be conferred by a naturally occurring gene introduced into thegenome of a line by backcrossing, a natural or induced mutation, or atransgene introduced through genetic transformation techniques into theplant or a progenitor of any previous generation thereof. Whenintroduced through transformation, a genetic locus may comprise one ormore genes integrated at a single chromosomal location.

The invention also concerns the seed of spinach hybrid RX 06681616and/or spinach lines SSB66-1090 F and SSB66-1131 M. The spinach seed ofthe invention may be provided as an essentially homogeneous populationof spinach seed of spinach hybrid RX 06681616 and/or spinach linesSSB66-1090 F and SSB66-1131 M. Essentially homogeneous populations ofseed are generally free from substantial numbers of other seed.Therefore, seed of hybrid RX 06681616 and/or spinach lines SSB66-1090 Fand SSB66-1131 M may be defined as forming at least about 97% of thetotal seed, including at least about 98%, 99% or more of the seed. Theseed population may be separately grown to provide an essentiallyhomogeneous population of spinach plants designated RX 06681616 and/orspinach lines SSB66-1090 F and SSB66-1131 M.

In yet another aspect of the invention, a tissue culture of regenerablecells of a spinach plant of hybrid RX 06681616 and/or spinach linesSSB66-1090 F and SSB66-1131 M is provided. The tissue culture willpreferably be capable of regenerating spinach plants capable ofexpressing all of the physiological and morphological characteristics ofthe starting plant, and of regenerating plants having substantially thesame genotype as the starting plant. Examples of some of thephysiological and morphological characteristics of the hybrid RX06681616 and/or spinach lines SSB66-1090 F and SSB66-1131 M includethose traits set forth in the tables herein. The regenerable cells insuch tissue cultures may be derived, for example, from embryos,meristems, cotyledons, pollen, leaves, anthers, roots, root tips,pistils, flowers, seed and stalks. Still further, the present inventionprovides spinach plants regenerated from a tissue culture of theinvention, the plants having all the physiological and morphologicalcharacteristics of hybrid RX 06681616 and/or spinach lines SSB66-1090 Fand SSB66-1131 M.

In still yet another aspect of the invention, processes are provided forproducing spinach seeds, plants and fruit, which processes generallycomprise crossing a first parent spinach plant with a second parentspinach plant, wherein at least one of the first or second parentspinach plants is a plant of spinach line SSB66-1090 F or spinach lineSSB66-1131 M. These processes may be further exemplified as processesfor preparing hybrid spinach seed or plants, wherein a first spinachplant is crossed with a second spinach plant of a different, distinctgenotype to provide a hybrid that has, as one of its parents, a plant ofspinach line SSB66-1090 F or spinach line SSB66-1131 M. In theseprocesses, crossing will result in the production of seed. The seedproduction occurs regardless of whether the seed is collected or not.

In one embodiment of the invention, the first step in “crossing”comprises planting seeds of a first and second parent spinach plant,often in proximity so that pollination will occur for example, mediatedby insect vectors. Alternatively, pollen can be transferred manually.Where the plant is self-pollinated, pollination may occur without theneed for direct human intervention other than plant cultivation.

A second step may comprise cultivating or growing the seeds of first andsecond parent spinach plants into plants that bear flowers. A third stepmay comprise preventing self-pollination of the plants, such as byemasculating the flowers (i.e., killing or removing the pollen).

A fourth step for a hybrid cross may comprise cross-pollination betweenthe first and second parent spinach plants. Yet another step comprisesharvesting the seeds from at least one of the parent spinach plants. Theharvested seed can be grown to produce a spinach plant or hybrid spinachplant.

The present invention also provides the spinach seeds and plantsproduced by a process that comprises crossing a first parent spinachplant with a second parent spinach plant, wherein at least one of thefirst or second parent spinach plants is a plant of spinach hybrid RX06681616 and/or spinach lines SSB66-1090 F and SSB66-1131 M. In oneembodiment of the invention, spinach seed and plants produced by theprocess are first generation (F₁) hybrid spinach seed and plantsproduced by crossing a plant in accordance with the invention withanother, distinct plant. The present invention further contemplatesplant parts of such an F₁ hybrid spinach plant, and methods of usethereof. Therefore, certain exemplary embodiments of the inventionprovide an F₁ hybrid spinach plant and seed thereof.

In still yet another aspect, the present invention provides a method ofproducing a plant derived from hybrid RX 06681616 and/or spinach linesSSB66-1090 F and SSB66-1131 M, the method comprising the steps of: (a)preparing a progeny plant derived from hybrid RX 06681616 and/or spinachlines SSB66-1090 F and SSB66-1131 M, wherein said preparing comprisescrossing a plant of the hybrid RX 06681616 and/or spinach linesSSB66-1090 F and SSB66-1131 M with a second plant; and (b) crossing theprogeny plant with itself or a second plant to produce a seed of aprogeny plant of a subsequent generation. In further embodiments, themethod may additionally comprise: (c) growing a progeny plant of asubsequent generation from said seed of a progeny plant of a subsequentgeneration and crossing the progeny plant of a subsequent generationwith itself or a second plant; and repeating the steps for an additional3-10 generations to produce a plant derived from hybrid RX 06681616and/or spinach lines SSB66-1090 F and SSB66-1131 M. The plant derivedfrom hybrid RX 06681616 and/or spinach lines SSB66-1090 F and SSB66-1131M may be an inbred line, and the aforementioned repeated crossing stepsmay be defined as comprising sufficient inbreeding to produce the inbredline. In the method, it may be desirable to select particular plantsresulting from step (c) for continued crossing according to steps (b)and (c). By selecting plants having one or more desirable traits, aplant derived from hybrid RX 06681616 and/or spinach lines SSB66-1090 Fand SSB66-1131 M is obtained which possesses some of the desirabletraits of the line/hybrid as well as potentially other selected traits.

In certain embodiments, the present invention provides a method ofproducing food or feed comprising: (a) obtaining a plant of spinachhybrid RX 06681616 and/or spinach lines SSB66-1090 F and SSB66-1131 M,wherein the plant has been cultivated to maturity, and (b) collecting atleast one spinach from the plant.

In still yet another aspect of the invention, the genetic complement ofspinach hybrid RX 06681616 and/or spinach lines SSB66-1090 F andSSB66-1131 M is provided. The phrase “genetic complement” is used torefer to the aggregate of nucleotide sequences, the expression of whichsequences defines the phenotype of, in the present case, a spinachplant, or a cell or tissue of that plant. A genetic complement thusrepresents the genetic makeup of a cell, tissue or plant, and a hybridgenetic complement represents the genetic make up of a hybrid cell,tissue or plant. The invention thus provides spinach plant cells thathave a genetic complement in accordance with the spinach plant cellsdisclosed herein, and seeds and plants containing such cells.

Plant genetic complements may be assessed by genetic marker profiles,and by the expression of phenotypic traits that are characteristic ofthe expression of the genetic complement, e.g., isozyme typing profiles.It is understood that hybrid RX 06681616 and/or spinach lines SSB66-1090F and SSB66-1131 M could be identified by any of the many well knowntechniques such as, for example, Simple Sequence Length Polymorphisms(SSLPs) (Williams et al., Nucleic Acids Res., 1 8:6531-6535, 1990),Randomly Amplified Polymorphic DNAs (RAPDs), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Arbitrary Primed Polymerase Chain Reaction (AP-PCR), Amplified FragmentLength Polymorphisms (AFLPs) (EP 534 858, specifically incorporatedherein by reference in its entirety), and Single NucleotidePolymorphisms (SNPs) (Wang et al., Science, 280:1077-1082, 1998).

In still yet another aspect, the present invention provides hybridgenetic complements, as represented by spinach plant cells, tissues,plants, and seeds, formed by the combination of a haploid geneticcomplement of a spinach plant of the invention with a haploid geneticcomplement of a second spinach plant, preferably, another, distinctspinach plant. In another aspect, the present invention provides aspinach plant regenerated from a tissue culture that comprises a hybridgenetic complement of this invention.

Any embodiment discussed herein with respect to one aspect of theinvention applies to other aspects of the invention as well, unlessspecifically noted.

The term “about” is used to indicate that a value includes the standarddeviation of the mean for the device or method being employed todetermine the value. The use of the term “or” in the claims is used tomean “and/or” unless explicitly indicated to refer to alternatives onlyor the alternatives are mutually exclusive. When used in conjunctionwith the word “comprising” or other open language in the claims, thewords “a” and “an” denote “one or more,” unless specifically notedotherwise. The terms “comprise,” “have” and “include” are open-endedlinking verbs. Any forms or tenses of one or more of these verbs, suchas “comprises,” “comprising,” “has,” “having,” “includes” and“including,” are also open-ended. For example, any method that“comprises,” “has” or “includes” one or more steps is not limited topossessing only those one or more steps and also covers other unlistedsteps. Similarly, any plant that “comprises,” “has” or “includes” one ormore traits is not limited to possessing only those one or more traitsand covers other unlisted traits.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and any specificexamples provided, while indicating specific embodiments of theinvention, are given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art from this detaileddescription.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions relating to plants,seeds and derivatives of spinach hybrid RX 06681616, spinach lineSSB66-1090 F and spinach line SSB66-1131 M.

Spinach (Spinacia oleracea L.) hybrid RX 06681616 with a smooth leavetype exhibits a number of beneficial traits including resistance toPeronospora farinose f.sp. spinaciae against the races Pfs 1 till 13 andagainst new isolate UA4410.

Spinach (Spinacia oleracea L.) line SSB66-1131 M exhibits a number ofbeneficial traits including resistance to Peronospora farinosa fsp.spinaciae against the races Pfs 1 till Pfs 13. The development of theline can be summarized as follows.

Spinach (Spinacia oleracea L.) line SSB66-1090 F exhibits a number ofbeneficial traits including resistance to downy mildew (Peronosporafarinosa f.sp. spinaciae (Pfs)) races Pfs 1,3,5,8,11,12 and the newdowny mildew isolate UA4410. The development of the line can besummarized as follows.

A. Origin and Breeding History of Spinach Hybrid RX 06681616

The parents of hybrid RX 06681616 are SSB66-1090 F and SSB66-1131 M.These parents were created as follows:

Line SSB66-1131 M was developed at the Monsanto Research station inWageningen, The Netherlands, by pedigree selection out of a breedingcross between F1[Trentino] Pop Vriend and inbred line SSB66-8M. Theorigin and selections that led to the development of SSB66-1131 M can besummarized as follows (S=Selfing, M=Mass selection):

Year Generation Material Main selection criteria Year 1 F1 [Trentino] *Making breeding cross SSB66-0008M Year 2 F2 Select for Pf resistance andplant type Year 3 F2.S1 Select for plant type (bolting) Year 3 F2.S2Uniformity Year 6 F2, S2.M1 Uniformity

The final outcome is a line, which combines a late bolting plant typewith resistances against the Downey mildew races Pfs 1 till Pfs 13.Observation during the mass selection one year and stock seed productionanother year confirmed that SSB66-1131 M is uniform and stable. As istrue with other spinach varieties, a small percentage of off-types canoccur for almost any characteristics during the course of repeatedmultiplications. However, no variants were observed during the two yearsin which SSB66-1131 M was observed to be uniform and stable.

SSB66-1090 F was developed at the Monsanto Research station inWageningen, The Netherlands, by pedigree selection out of a breedingcross in which we wanted to combine good plant characteristics from theinbred line Movak (Monsanto) with desired downy mildew resistances fromthe hybrid Spartacus (Bejo). The origin and selections that led to thedevelopment of SSB66-1090 F can be summarized as follows (S=Selfing,M=Mass selection):

Year Generation Material Main selection criteria Year 1 F1 [Spartacus *Movak] Breeding cross Year 4 F2 Pfs resistance Year 5 F2.1. Pfs res.,color, productivity Year 6 F2.2. Pfs res., color, productivity Year 7F2.2.S1 Pfs res., color, productivity, uniformity Year 8 F2.2.S2 color,productivity, uniformity Year 9 F2.2.S3 Uniformity Year 12 F2.2.S3.M1Uniformity Year 15 F2.2.S3.M2 Uniformity

The final outcome is a line, which combines a good color with theresistance against the races Pfs1, 3, 5, 8, 12 and the new downy mildewisolate UA4410. Observation during the Mass selection one year and thestock seed production in another year confirmed that SSB66-1090 F isuniform and stable. As is true with other spinach varieties, a smallpercentage of off-types can occur for almost any characteristics duringthe course of repeated multiplications. However, no variants wereobserved during the two years in which SSB66-1090 F was observed to beuniform and stable.

The parent lines are uniform and stable, as is a hybrid producedtherefrom. A small percentage of variants can occur within commerciallyacceptable limits for almost any characteristic during the course ofrepeated multiplication. However no variants are expected.

B. Physiological and Morphological Characteristics of Spinach Hybrid RX06681616, Spinach Line SSB66-1090 F and Spinach Line SSB66-1131 M

In accordance with one aspect of the present invention, there isprovided a plant having the physiological and morphologicalcharacteristics of spinach hybrid RX 06681616 and the parent linesthereof. A description of the physiological and morphologicalcharacteristics of such plants is presented in Tables 1-3.

TABLE 1 Physiological and Morphological Characteristics of Hybrid RX06681616 Comparison Variety: CHARACTERISTIC RX 06681616 Chica Ploidydiploid diploid Maturity Growth Rate slow (Norgreen) slow (Norgreen)days from planting to prime 45 45 market stage Plant (Prime MarketStage) Habit flat (Viroflay) flat (Viroflay) Size medium medium Spread(cm) 47 cm 49 cm Height (cm) 13 cm 14 cm Seedling Cotyledon Length ofcotyledon medium medium Width (mm) 5.2 mm 6.9 mm Length (mm) 57 mm 54 mmTip pointed rounded Color medium green medium green Color Chart Name RHSColor Chart Value 143A 146B Leaf (First Foliage Leaves) Shape ovateovate Base V-shape V-shape Tip round-pointed round-pointed Marginslightly curled curled under Upper Surface Color medium green (GiantNobel) medium green (Giant Nobel) Color Chart Name RHS Color Chart Value146A 146A Lower Surface Color lighter lighter (Compared with uppersurface) Color Chart Name RHS Color Chart Value 146B 146B Leaf (PrimeMarket Stage) Surface smooth (Viroflay) smooth (Viroflay) Base lobedlobed Tip round round Margin slightly curved slightly curved UpperSurface Color dark green (Standing dark green (Standing Bloomsdale)Bloomsdale) Color Chart Name RHS Color Chart Value 146A+ 146A+ LowerSurface Color lighter lighter (Compared with upper surface) Color ChartName RHS Color Chart Value 146B 146B Luster glossy glossy Blade sizelarge (Giant Nobel) large (Giant Nobel) Blade Intensity of green colordark [Imola, Lavewa, Nores] dark [Imola, Lavewa, Nores] Blade Blisteringmedium [Butterflay, Koala, medium [Butterflay, Koala, Mystic] Mystic]Blade Lobing absent or very weak (US = not absent or very weak (US = notlobed) lobed) Blade Attitude horizontal [Lavewa, Mystic] horizontal[Lavewa, Mystic] Blade Shape [excluding basal broad ovate broad ovatelobes] (prime market stage) Blade curving of margin recurved (Imola)recurved (Imola) Blade shape of apex rounded (Imola, Nores) rounded(Imola, Nores) Blade shape in longitudinal convex (Grappa, Lazio) convex(Grappa, Lazio) section Petiole Attitude horizontal [Comte, Lavewa]horizontal [Comte, Lavewa] Petiole Length (prime market medium [TG =Butterflay, medium [TG = Butterflay, stage) Giraffe] Giraffe] PetioleColor light green light green Color Chart Name RHS Color Chart Value146B 146B Petiole Red Pigmentation absent absent Petiole Length to theBlade 8 cm 8 cm Petiole Diameter (mm) 7.2 mm 8.0 mm Petiole Diameterlarge (Giant Nobel) large (Giant Nobel) Seed Stalk Development Start ofBolting (10% of late (Norgreen) late (Norgreen) plants) Time of start ofbolting (for very late [Chico, Lavewa] very late [Chico, Lavewa] springsown crop, 15% of plants) Height of Stalk (cm) 60 cm 57 cm Leaves onStalk of Female many many Plant Leaves on Stalk of Male many many PlantPlants that are Female 0-10% [Monnopa] 0-10% [Monnopa] Plants that areMale 0-10% [Monnopa, Parrot] 0-10% [Monnopa, Parrot] Plants that areMonoecious 91-100% [Monnopa] 91-100% [Monnopa] Seed Surface smoothsmooth Spines (harvested seed) absent [Resistoflay] absent [Resistoflay]*These are typical values. Values may vary due to environment. Othervalues that are substantially equivalent are also within the scope ofthe invention.

TABLE 2 Physiological and Morphological Characteristics of LineSSB66-1090 F CHARACTERISTIC SSB-66-1090F SSB-66-1042F Ploidy diploiddiploid Maturity Growth Rate medium (Long Standing medium (Long StandingBloomsdale) Bloomsdale) Days from planting to prime 43 43 market stagePlant (Prime Market Stage) Habit semi-erect (Long Standing semi-erect(Long Standing Bloomsdale) Bloomsdale) Size medium medium Spread (cm) 44cm 43 cm Height (cm) 13 cm 13 cm Seedling Cotyledon Length of cotyledonshort [Nores] medium Width (mm) 5.2 mm 5.3 mm Length (mm) 62 mm 60 mmTip pointed rounded Color medium green medium green Color Chart Name RHSColor Chart Value 146B 146B Leaf (First Foliage Leaves) Shape circularcircular Base V-shape V-shape Tip round round Margin slightly curledslightly curled Upper Surface Color dark green (Long Standing dark green(Long Standing Bloomsdale) Bloomsdale) Color Chart Name RHS Color ChartValue 147A 147A Lower Surface Color lighter lighter (Compared with uppersurface) Color Chart Name RHS Color Chart Value 147B 147B Leaf (PrimeMarket Stage) Surface smooth (Viroflay) smooth (Viroflay) Base lobedlobed Tip round round Margin curled under curled under Upper SurfaceColor dark green (Standing dark green (Standing Bloomsdale) Bloomsdale)Color Chart Name RHS Color Chart Value 147A 147A Lower Surface Colorlighter lighter (Compared with upper surface) Color Chart Name RHS ColorChart Value 146B 146B Luster glossy glossy Blade size medium (VirginiaSavoy) medium (Virginia Savoy) Blade Blistering medium [Butterflay,Koala, medium [Butterflay, Koala, Mystic] Mystic] Blade Lobing absent orvery weak (US = not weak [Butterflay, Giraffe] lobed) Blade Attitudehorizontal [Lavewa, Mystic] semi-pendulous [Giraffe, Medania] BladeShape [excluding basal broad ovate broad ovate lobes] (prime marketstage) Blade curving of margin recurved (Imola) recurved (Imola) Bladeshape of apex rounded (Imola, Nores) rounded (Imola, Nores) Blade shapein longitudinal convex (Grappa, Lazio) convex (Grappa, Lazio) sectionPetiole Attitude horizontal [Comte, Lavewa] horizontal [Comte, Lavewa]Petiole Color light green light green Color Chart Name RHS Color ChartValue 146C 146C Petiole Red Pigmentation absent absent Petiole Length tothe Blade 8 cm 12 cm Petiole Diameter (mm) 8 mm 6 mm Petiole Diameterlarge (Giant Nobel) medium Seed Stalk Development Start of Bolting (10%of late (Norgreen) late (Norgreen) plants) Time of start of bolting (forvery late [Chico, Lavewa] very late [Chico, Lavewa] spring sown crop,15% of plants) Height of Stalk (cm) 71 cm 75 cm Leaves on Stalk ofFemale many many Plant leaves on stalk of male plant many many Plantsthat are Female 0-10% [Monnopa] 0-10% [Monnopa] Plants that are Male0-10% [Monnopa, Parrot] 0-10% [Monnopa, Parrot] Plants that areMonoecious 91-100% [Monnopa] 91-100% [Monnopa] Seed Surface smoothsmooth Spines (harvested seed) absent [Resistoflay] absent [Resistoflay]*These are typical values. Values may vary due to environment. Othervalues that are substantially equivalent are also within the scope ofthe invention.

TABLE 3 Physiological and Morphological Characteristics of LineSSB66-1131 M Comparison Variety: CHARACTERISTIC SSB 66-1131M SSB66-1061M Ploidy diploid diploid Maturity Growth Rate slow (Norgreen)medium (Long Standing Bloomsdale) Days from planting to prime 47 44market stage Plant (Prime Market Stage) Habit flat (Viroflay) flat(Viroflay) Size small (America) medium Spread (cm) 35 cm 45 cm Height(cm) 8 cm 14 cm Seedling Cotyledon Length of cotyledon short [Nores]medium Width (mm) 4.3 mm 4.7 mm Length (mm) 35 mm 57 mm Tip pointedpointed Color dark green medium green Color Chart Name RHS Color ChartValue 146A 146B Leaf (First Foliage Leaves) Shape ovate ovate Basestraight straight Tip round-pointed round-pointed Margin slightly curledslightly curled Upper Surface Color medium green (Giant Nobel) mediumgreen (Giant Nobel) Color Chart Name RHS Color Chart Value 147A 147ALower Surface Color lighter lighter (Compared with upper surface) ColorChart Name RHS Color Chart Value 147B 147B Leaf (Prime Market Stage)Surface smooth (Viroflay) smooth (Viroflay) Base lobed lobed Tip roundround Margin curled under slightly curved Upper Surface Color mediumgreen (Giant Nobel) medium green (Giant Nobel) Color Chart Name RHSColor Chart Value 147A 146A Lower Surface Color lighter lighter(Compared with upper surface) Color Chart Name RHS Color Chart Value146A 146B Luster dull glossy Blade size small (Long Standing medium(Virginia Savoy) Bloomsdale) Blade Blistering weak [Polka, Tarpy] medium[Butterflay, Koala, Mystic] Blade Lobing absent or very weak (US =absent or very weak (US = not lobed) not lobed) Blade Attitudehorizontal [Lavewa, Mystic] semi-erect [Grappa, Monnopa] Blade Shape[excluding basal broad ovate broad ovate lobes] (prime market stage)Blade curving of margin recurved (Imola) recurved (Imola) Blade shape ofapex rounded (Imola, Nores) rounded (Imola, Nores) Blade shape inlongitudinal flat (Mystic, Resistoflay) flat (Mystic, Resistoflay)section Petiole Attitude horizontal [Comte, Lavewa] horizontal [Comte,Lavewa] Petiole Length (prime market short [TG = Imola, Mystic] medium[TG = Butterflay, stage) Giraffe] Petiole Color light green light greenColor Chart Name RHS Color Chart Value 143A 143A Petiole RedPigmentation absent absent Petiole Length to the Blade 4 cm 9 cm PetioleDiameter (mm) 6 mm 7 mm Petiole Diameter medium medium Seed StalkDevelopment Start of Bolting (10% of late (Norgreen) late (Norgreen)plants) Time of start of bolting (for very late [Chico, Lavewa] verylate [Chico, Lavewa] spring sown crop, 15% of plants) Height of Stalk(cm) 48 cm 56 cm Leaves on Stalk of Female many Plant Leaves on Stalk ofMale many many Plant Plants that are Female 0-10% [Monnopa] 0-10%[Monnopa] Plants that are Male 0-10% [Monnopa, Parrot] 0-10% [Monnopa,Parrot] Plants that are Monoecious 91-100% [Monnopa] 91-100% [Monnopa]Seed Surface smooth smooth Spines (harvested seed) absent [Resistoflay]absent [Resistoflay] *These are typical values. Values may vary due toenvironment. Other values that are substantially equivalent are alsowithin the scope of the invention.

C. Breeding Spinach Plants

One aspect of the current invention concerns methods for producing seedof spinach hybrid RX 06681616 involving crossing spinach linesSSB66-1090 F and SSB66-1131 M. Alternatively, in other embodiments ofthe invention, hybrid RX 06681616, line SSB66-1090 F, or line SSB66-1131M may be crossed with itself or with any second plant. Such methods canbe used for propagation of hybrid RX 06681616 and/or the spinach linesSSB66-1090 F and SSB66-1131 M, or can be used to produce plants that arederived from hybrid RX 06681616 and/or the spinach lines SSB66-1090 Fand SSB66-1131 M. Plants derived from hybrid RX 06681616 and/or thespinach lines SSB66-1090 F and SSB66-1131 M may be used, in certainembodiments, for the development of new spinach varieties.

The development of new varieties using one or more starting varieties iswell known in the art. In accordance with the invention, novel varietiesmay be created by crossing hybrid RX 06681616 followed by multiplegenerations of breeding according to such well known methods. Newvarieties may be created by crossing with any second plant. In selectingsuch a second plant to cross for the purpose of developing novel lines,it may be desired to choose those plants which either themselves exhibitone or more selected desirable characteristics or which exhibit thedesired characteristic(s) when in hybrid combination. Once initialcrosses have been made, inbreeding and selection take place to producenew varieties. For development of a uniform line, often five or moregenerations of selfing and selection are involved.

Uniform lines of new varieties may also be developed by way ofdouble-haploids. This technique allows the creation of true breedinglines without the need for multiple generations of selfing andselection. In this manner true breeding lines can be produced in aslittle as one generation. Haploid embryos may be produced frommicrospores, pollen, anther cultures, or ovary cultures. The haploidembryos may then be doubled autonomously, or by chemical treatments(e.g. colchicine treatment). Alternatively, haploid embryos may be growninto haploid plants and treated to induce chromosome doubling. In eithercase, fertile homozygous plants are obtained. In accordance with theinvention, any of such techniques may be used in connection with a plantof the invention and progeny thereof to achieve a homozygous line.

Backcrossing can also be used to improve an inbred plant. Backcrossingtransfers a specific desirable trait from one inbred or non-inbredsource to an inbred that lacks that trait. This can be accomplished, forexample, by first crossing a superior inbred (A) (recurrent parent) to adonor inbred (non-recurrent parent), which carries the appropriate locusor loci for the trait in question. The progeny of this cross are thenmated back to the superior recurrent parent (A) followed by selection inthe resultant progeny for the desired trait to be transferred from thenon-recurrent parent. After five or more backcross generations withselection for the desired trait, the progeny have the characteristicbeing transferred, but are like the superior parent for most or almostall other loci. The last backcross generation would be selfed to givepure breeding progeny for the trait being transferred.

The plants of the present invention are particularly well suited for thedevelopment of new lines based on the elite nature of the geneticbackground of the plants. In selecting a second plant to cross with RX06681616 and/or spinach lines SSB66-1090 F and SSB66-1131 M for thepurpose of developing novel spinach lines, it will typically bepreferred to choose those plants which either themselves exhibit one ormore selected desirable characteristics or which exhibit the desiredcharacteristic(s) when in hybrid combination. Examples of desirabletraits may include, in specific embodiments, high seed yield, high seedgermination, seedling vigor, high fruit yield, disease tolerance orresistance, and adaptability for soil and climate conditions.Consumer-driven traits, such as a fruit shape, color, texture, and tasteare other examples of traits that may be incorporated into new lines ofspinach plants developed by this invention.

D. Performance Characteristics

As described above, hybrid RX 06681616 exhibits desirable traits, asconferred by spinach lines SSB66-1090 F and SSB66-1131 M. Theperformance characteristics of hybrid RX 06681616 and spinach linesSSB66-1090 F and SSB66-1131 M were the subject of an objective analysisof the performance traits relative to other varieties. The results ofthe analysis are presented below.

Spinach line SSB66-1090 F can be characterized as a late bolting linethat has spineless seeds (smooth), with dark color and with a resistanceto downy mildew (Peronospora farinose f.sp. spinaciae (Pfs) races Pfs 1,3, 5, 8, 11, 12 and the new Downey mildew isolate UA4410.

The parental line believed to most closely resemble SSB66-1090 F isMonsanto parental line SSB66-1042F. Comparative characteristicsdistinguish the two lines that include, but may not be limited tobolting, petiole length and resistances. The candidate line SSB66-1090 Fis later bolting, has got longer petiole length and other resistances(see table below) compared to SSB66-1042M.

Spinach line SSB66-1131 M can be characterized as a late bolting linethat has spineless seeds (smooth) and with a resistance to downy mildew(Peronospora farinosa fsp. spinaciae (Pfs) races Pfs 1 till Pfs 13.Furthermore the line is phenotypically distinct from all well-knownmaterial by a combination of dark and small leafs.

The parental line believed to most closely resemble SSB66-1131 M isMonsanto parental line SSB66-1061M. Comparative characteristicsdistinguish the two lines that include, but may not be limited toresistances, leaf shape, leaf color and seed stalk length. The candidateline SSB66-1131 M has other more resistances, has rounder leaf shape,has got darker color and shorter seed stalk length compared toSMB66-1061M.

TABLE 4 Performance Characteristics for Hybrid RX 06681616 andComparative varieties Material Seed stalk Resistances RX 06681616 MediumPfs 1-13 + UA4410 Chica Short Pfs 1-4

TABLE 5 Performance Characteristics For Line SSB66- 1090 F andComparative Variety SSB66-1042F Petiole Bolting Length (cm) Material2007 2008 2009 2010 2011 2011 Resistances SSB66- 37 39 43 46 39 12 Pfs1, 3, 5, 1090F 8, 11, 12, UA 4410 SSB66- 36 35 34 42 35 6 Pfs 1, 2, 3,1042F 4

TABLE 6 Performance Characteristics For Line SSB66-1131 M andComparative Variety SSB66-1061M Seed stalk Leaf length Material Leafcolor shape (cm) Resistances Pfs SSB66-1061M Medium dark avate 56 1until 7, 9, 11, 13 SSB66-1131 M dark Round 48 1 until 13

E. Further Embodiments of the Invention

In certain aspects of the invention, plants described herein areprovided modified to include at least a first desired heritable trait.Such plants may, in one embodiment, be developed by a plant breedingtechnique called backcrossing, wherein essentially all of themorphological and physiological characteristics of a variety arerecovered in addition to a genetic locus transferred into the plant viathe backcrossing technique. The term single locus converted plant asused herein refers to those spinach plants which are developed by aplant breeding technique called backcrossing, wherein essentially all ofthe morphological and physiological characteristics of a variety arerecovered in addition to the single locus transferred into the varietyvia the backcrossing technique. By essentially all of the morphologicaland physiological characteristics, it is meant that the characteristicsof a plant are recovered that are otherwise present when compared in thesame environment, other than an occasional variant trait that mightarise during backcrossing or direct introduction of a transgene.

Backcrossing methods can be used with the present invention to improveor introduce a characteristic into the present variety. The parentalspinach plant which contributes the locus for the desired characteristicis termed the nonrecurrent or donor parent. This terminology refers tothe fact that the nonrecurrent parent is used one time in the backcrossprotocol and therefore does not recur. The parental spinach plant towhich the locus or loci from the nonrecurrent parent are transferred isknown as the recurrent parent as it is used for several rounds in thebackcrossing protocol.

In a typical backcross protocol, the original variety of interest(recurrent parent) is crossed to a second variety (nonrecurrent parent)that carries the single locus of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a spinach plant isobtained wherein essentially all of the morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, in addition to the single transferred locus from the nonrecurrentparent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single locus of the recurrent variety ismodified or substituted with the desired locus from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphologicalconstitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially desirable trait to theplant. The exact backcrossing protocol will depend on the characteristicor trait being altered and the genetic distance between the recurrentand nonrecurrent parents. Although backcrossing methods are simplifiedwhen the characteristic being transferred is a dominant allele, arecessive allele, or an additive allele (between recessive anddominant), may also be transferred. In this instance it may be necessaryto introduce a test of the progeny to determine if the desiredcharacteristic has been successfully transferred.

In one embodiment, progeny spinach plants of a backcross in which aplant described herein is the recurrent parent comprise (i) the desiredtrait from the non-recurrent parent and (ii) all of the physiologicaland morphological characteristics of spinach the recurrent parent asdetermined at the 5% significance level when grown in the sameenvironmental conditions.

New varieties can also be developed from more than two parents. Thetechnique, known as modified backcrossing, uses different recurrentparents during the backcrossing. Modified backcrossing may be used toreplace the original recurrent parent with a variety having certain moredesirable characteristics or multiple parents may be used to obtaindifferent desirable characteristics from each.

With the development of molecular markers associated with particulartraits, it is possible to add additional traits into an established germline, such as represented here, with the end result being substantiallythe same base germplasm with the addition of a new trait or traits.Molecular breeding, as described in Moose and Mumm, 2008 (PlantPhysiology, 147: 969-977), for example, and elsewhere, provides amechanism for integrating single or multiple traits or QTL into an eliteline. This molecular breeding-facilitated movement of a trait or traitsinto an elite line may encompass incorporation of a particular genomicfragment associated with a particular trait of interest into the eliteline by the mechanism of identification of the integrated genomicfragment with the use of flanking or associated marker assays. In theembodiment represented here, one, two, three or four genomic loci, forexample, may be integrated into an elite line via this methodology. Whenthis elite line containing the additional loci is further crossed withanother parental elite line to produce hybrid offspring, it is possibleto then incorporate at least eight separate additional loci into thehybrid. These additional loci may confer, for example, such traits as adisease resistance or a fruit quality trait. In one embodiment, eachlocus may confer a separate trait. In another embodiment, loci may needto be homozygous and exist in each parent line to confer a trait in thehybrid. In yet another embodiment, multiple loci may be combined toconfer a single robust phenotype of a desired trait.

Many single locus traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single locus traits may or may not betransgenic; examples of these traits include, but are not limited to,herbicide resistance, resistance to bacterial, fungal, or viral disease,insect resistance, modified fatty acid or carbohydrate metabolism, andaltered nutritional quality. These comprise genes generally inheritedthrough the nucleus.

Direct selection may be applied where the single locus acts as adominant trait. For this selection process, the progeny of the initialcross are assayed for viral resistance and/or the presence of thecorresponding gene prior to the backcrossing. Selection eliminates anyplants that do not have the desired gene and resistance trait, and onlythose plants that have the trait are used in the subsequent backcross.This process is then repeated for all additional backcross generations.

Selection of spinach plants for breeding is not necessarily dependent onthe phenotype of a plant and instead can be based on geneticinvestigations. For example, one can utilize a suitable genetic markerwhich is closely genetically linked to a trait of interest. One of thesemarkers can be used to identify the presence or absence of a trait inthe offspring of a particular cross, and can be used in selection ofprogeny for continued breeding. This technique is commonly referred toas marker assisted selection. Any other type of genetic marker or otherassay which is able to identify the relative presence or absence of atrait of interest in a plant can also be useful for breeding purposes.Procedures for marker assisted selection are well known in the art. Suchmethods will be of particular utility in the case of recessive traitsand variable phenotypes, or where conventional assays may be moreexpensive, time consuming or otherwise disadvantageous. Types of geneticmarkers which could be used in accordance with the invention include,but are not necessarily limited to, Simple Sequence Length Polymorphisms(SSLPs) (Williams et al., Nucleic Acids Res., 1 8:6531-6535, 1990),Randomly Amplified Polymorphic DNAs (RAPDs), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Arbitrary Primed Polymerase Chain Reaction (AP-PCR), Amplified FragmentLength Polymorphisms (AFLPs) (EP 534 858, specifically incorporatedherein by reference in its entirety), and Single NucleotidePolymorphisms (SNPs) (Wang et al., Science, 280:1077-1082, 1998).

F. Plants Derived by Genetic Engineering

Many useful traits that can be introduced by backcrossing, as well asdirectly into a plant, are those which are introduced by genetictransformation techniques. Genetic transformation may therefore be usedto insert a selected transgene into a plant of the invention or may,alternatively, be used for the preparation of transgenes which can beintroduced by backcrossing. Methods for the transformation of plantsthat are well known to those of skill in the art and applicable to manycrop species include, but are not limited to, electroporation,microprojectile bombardment, Agrobacterium-mediated transformation anddirect DNA uptake by protoplasts.

To effect transformation by electroporation, one may employ eitherfriable tissues, such as a suspension culture of cells or embryogeniccallus or alternatively one may transform immature embryos or otherorganized tissue directly. In this technique, one would partiallydegrade the cell walls of the chosen cells by exposing them topectin-degrading enzymes (pectolyases) or mechanically wound tissues ina controlled manner.

An efficient method for delivering transforming DNA segments to plantcells is microprojectile bombardment. In this method, particles arecoated with nucleic acids and delivered into cells by a propellingforce. Exemplary particles include those comprised of tungsten,platinum, and preferably, gold. For the bombardment, cells in suspensionare concentrated on filters or solid culture medium. Alternatively,immature embryos or other target cells may be arranged on solid culturemedium. The cells to be bombarded are positioned at an appropriatedistance below the macroprojectile stopping plate.

An illustrative embodiment of a method for delivering DNA into plantcells by acceleration is the Biolistics Particle Delivery System, whichcan be used to propel particles coated with DNA or cells through ascreen, such as a stainless steel or Nytex screen, onto a surfacecovered with target cells. The screen disperses the particles so thatthey are not delivered to the recipient cells in large aggregates.Microprojectile bombardment techniques are widely applicable, and may beused to transform virtually any plant species.

Agrobacterium-mediated transfer is another widely applicable system forintroducing gene loci into plant cells. An advantage of the technique isthat DNA can be introduced into whole plant tissues, thereby bypassingthe need for regeneration of an intact plant from a protoplast. ModernAgrobacterium transformation vectors are capable of replication in E.coli as well as Agrobacterium, allowing for convenient manipulations(Klee et al., Bio-Technology, 3(7):637-642, 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate the construction of vectors capable ofexpressing various polypeptide coding genes. The vectors described haveconvenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes. Additionally, Agrobacterium containing both armed anddisarmed Ti genes can be used for transformation.

In those plant strains where Agrobacterium-mediated transformation isefficient, it is the method of choice because of the facile and definednature of the gene locus transfer. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art (Fraley et al., Bio/Technology, 3:629-635, 1985; U.S.Pat. No. 5,563,055).

Transformation of plant protoplasts also can be achieved using methodsbased on calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see, e.g.,Potrykus et al., Mol. Gen. Genet., 199:183-188, 1985; Omirulleh et al.,Plant Mol. Biol., 21(3):415-428, 1993; Fromm et al., Nature,312:791-793, 1986; Uchimiya et al., Mol. Gen. Genet., 204:204, 1986;Marcotte et al., Nature, 335:454, 1988). Transformation of plants andexpression of foreign genetic elements is exemplified in Choi et al.(Plant Cell Rep., 13: 344-348, 1994), and Ellul et al. (Theor. Appl.Genet., 107:462-469, 2003).

A number of promoters have utility for plant gene expression for anygene of interest including but not limited to selectable markers,scoreable markers, genes for pest tolerance, disease resistance,nutritional enhancements and any other gene of agronomic interest.Examples of constitutive promoters useful for plant gene expressioninclude, but are not limited to, the cauliflower mosaic virus (CaMV)P-35S promoter, which confers constitutive, high-level expression inmost plant tissues (see, e.g., Odel et al., Nature, 313:810, 1985),including in monocots (see, e.g., Dekeyser et al., Plant Cell, 2:591,1990; Terada and Shimamoto, Mol. Gen. Genet., 220:389, 1990); a tandemlyduplicated version of the CaMV 35S promoter, the enhanced 35S promoter(P-e35S);1 the nopaline synthase promoter (An et al., Plant Physiol.,88:547, 1988); the octopine synthase promoter (Fromm et al., Plant Cell,1:977, 1989); and the figwort mosaic virus (P-FMV) promoter as describedin U.S. Pat. No. 5,378,619 and an enhanced version of the FMV promoter(P-eFMV) where the promoter sequence of P-FMV is duplicated in tandem;the cauliflower mosaic virus 19S promoter; a sugarcane bacilliform viruspromoter; a commelina yellow mottle virus promoter; and other plant DNAvirus promoters known to express in plant cells.

A variety of plant gene promoters that are regulated in response toenvironmental, hormonal, chemical, and/or developmental signals can alsobe used for expression of an operably linked gene in plant cells,including promoters regulated by (1) heat (Callis et al., PlantPhysiol., 88:965, 1988), (2) light (e.g., pea rbcS-3A promoter,Kuhlemeier et al., Plant Cell, 1:471, 1989; maize rbcS promoter,Schaffner and Sheen, Plant Cell, 3:997, 1991; or chlorophyll a/b-bindingprotein promoter, Simpson et al., EMBO J., 4:2723, 1985), (3) hormones,such as abscisic acid (Marcotte et al., Plant Cell, 1:969, 1989), (4)wounding (e.g., wunl, Siebertz et al., Plant Cell, 1:961, 1989); or (5)chemicals such as methyl jasmonate, salicylic acid, or Safener. It mayalso be advantageous to employ organ-specific promoters (e.g., Roshal etal., EMBO J., 6:1155, 1987; Schernthaner et al., EMBO J., 7:1249, 1988;Bustos et al., Plant Cell, 1:839, 1989).

Exemplary nucleic acids which may be introduced to plants of thisinvention include, for example, DNA sequences or genes from anotherspecies, or even genes or sequences which originate with or are presentin the same species, but are incorporated into recipient cells bygenetic engineering methods rather than classical reproduction orbreeding techniques. However, the term “exogenous” is also intended torefer to genes that are not normally present in the cell beingtransformed, or perhaps simply not present in the form, structure, etc.,as found in the transforming DNA segment or gene, or genes which arenormally present and that one desires to express in a manner thatdiffers from the natural expression pattern, e.g., to over-express.Thus, the term “exogenous” gene or DNA is intended to refer to any geneor DNA segment that is introduced into a recipient cell, regardless ofwhether a similar gene may already be present in such a cell. The typeof DNA included in the exogenous DNA can include DNA which is alreadypresent in the plant cell, DNA from another plant, DNA from a differentorganism, or a DNA generated externally, such as a DNA sequencecontaining an antisense message of a gene, or a DNA sequence encoding asynthetic or modified version of a gene.

Many hundreds if not thousands of different genes are known and couldpotentially be introduced into a spinach plant according to theinvention. Non-limiting examples of particular genes and correspondingphenotypes one may choose to introduce into a spinach plant include oneor more genes for insect tolerance, such as a Bacillus thuringiensis(B.t.) gene, pest tolerance such as genes for fungal disease control,herbicide tolerance such as genes conferring glyphosate tolerance, andgenes for quality improvements such as yield, nutritional enhancements,environmental or stress tolerances, or any desirable changes in plantphysiology, growth, development, morphology or plant product(s). Forexample, structural genes would include any gene that confers insecttolerance including but not limited to a Bacillus insect control proteingene as described in WO 99/31248, herein incorporated by reference inits entirety, U.S. Pat. No. 5,689,052, herein incorporated by referencein its entirety, U.S. Pat. Nos. 5,500,365 and 5,880,275, hereinincorporated by reference in their entirety. In another embodiment, thestructural gene can confer tolerance to the herbicide glyphosate asconferred by genes including, but not limited to Agrobacterium strainCP4 glyphosate resistant EPSPS gene (aroA:CP4) as described in U.S. Pat.No. 5,633,435, herein incorporated by reference in its entirety, orglyphosate oxidoreductase gene (GOX) as described in U.S. Pat. No.5,463,175, herein incorporated by reference in its entirety.

Alternatively, the DNA coding sequences can affect these phenotypes byencoding a non-translatable RNA molecule that causes the targetedinhibition of expression of an endogenous gene, for example viaantisense- or cosuppression-mediated mechanisms (see, for example, Birdet al., Biotech. Gen. Engin. Rev., 9:207, 1991). The RNA could also be acatalytic RNA molecule (i.e., a ribozyme) engineered to cleave a desiredendogenous mRNA product (see for example, Gibson and Shillito, Mol.Biotech., 7:125,1997). Thus, any gene which produces a protein or mRNAwhich expresses a phenotype or morphology change of interest is usefulfor the practice of the present invention.

G. Definitions

In the description and tables herein, a number of terms are used. Inorder to provide a clear and consistent understanding of thespecification and claims, the following definitions are provided:

Allele: Any of one or more alternative forms of a gene locus, all ofwhich alleles relate to one trait or characteristic. In a diploid cellor organism, the two alleles of a given gene occupy corresponding locion a pair of homologous chromosomes.

Backcrossing: A process in which a breeder repeatedly crosses hybridprogeny, for example a first generation hybrid (F₁), back to one of theparents of the hybrid progeny. Backcrossing can be used to introduce oneor more single locus conversions from one genetic background intoanother.

Crossing: The mating of two parent plants.

Cross-pollination: Fertilization by the union of two gametes fromdifferent plants.

Diploid: A cell or organism having two sets of chromosomes.

Emasculate: The removal of plant male sex organs or the inactivation ofthe organs with a cytoplasmic or nuclear genetic factor or a chemicalagent conferring male sterility.

Enzymes: Molecules which can act as catalysts in biological reactions.

F₁ Hybrid: The first generation progeny of the cross of two nonisogenicplants.

Genotype: The genetic constitution of a cell or organism.

Haploid: A cell or organism having one set of the two sets ofchromosomes in a diploid.

Linkage: A phenomenon wherein alleles on the same chromosome tend tosegregate together more often than expected by chance if theirtransmission was independent.

Marker: A readily detectable phenotype, preferably inherited incodominant fashion (both alleles at a locus in a diploid heterozygoteare readily detectable), with no environmental variance component, i.e.,heritability of 1.

Phenotype: The detectable characteristics of a cell or organism, whichcharacteristics are the manifestation of gene expression.

Quantitative Trait Loci (QTL): Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Resistance: As used herein, the terms “resistance” and “tolerance” areused interchangeably to describe plants that show no symptoms to aspecified biotic pest, pathogen, abiotic influence or environmentalcondition. These terms are also used to describe plants showing somesymptoms but that are still able to produce marketable product with anacceptable yield. Some plants that are referred to as resistant ortolerant are only so in the sense that they may still produce a crop,even though the plants are stunted and the yield is reduced.

Regeneration: The development of a plant from tissue culture.

Royal Horticultural Society (RHS) color chart value: The RHS color chartis a standardized reference which allows accurate identification of anycolor. A color's designation on the chart describes its hue, brightnessand saturation. A color is precisely named by the RHS color chart byidentifying the group name, sheet number and letter, e.g., Yellow-OrangeGroup 19A or Red Group 41B.

Self-pollination: The transfer of pollen from the anther to the stigmaof the same plant.

Single Locus Converted (Conversion) Plant: Plants which are developed bya plant breeding technique called backcrossing, wherein essentially allof the morphological and physiological characteristics of a spinachvariety are recovered in addition to the characteristics of the singlelocus transferred into the variety via the backcrossing technique and/orby genetic transformation.

Substantially Equivalent: A characteristic that, when compared, does notshow a statistically significant difference (e.g., p=0.05) from themean.

Tissue Culture: A composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant.

Transgene: A genetic locus comprising a sequence which has beenintroduced into the genome of a spinach plant by transformation.

H. Deposit Information

A deposit of spinach hybrid RX 06681616 and inbred parent linesSSB66-1090 F and SSB66-1131 M, disclosed above and recited in theclaims, has been made with the American Type Culture Collection (ATCC),10801 University Blvd., Manassas, Va. 20110-2209. The date of depositwere Sep. 6, 2011, Aug. 2, 2011, and Aug. 19, 2011. The accessionnumbers for those deposited seeds of spinach hybrid RX 06681616 andinbred parent lines SSB66-1090 F and SSB66-1131 M are ATCC Accession No.PTA-12059, ATCC Accession No. PTA-12018, and ATCC Accession No.PTA-12041, respectively. Upon issuance of a patent, all restrictionsupon the deposits will be removed, and the deposits are intended to meetall of the requirements of 37 C.F.R. §1.801-1.809. The deposits will bemaintained in the depository for a period of 30 years, or 5 years afterthe last request, or for the effective life of the patent, whichever islonger, and will be replaced if necessary during that period.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the invention, as limited only bythe scope of the appended claims.

All references cited herein are hereby expressly incorporated herein byreference.

What is claimed is:
 1. A spinach plant comprising at least a first setof the chromosomes of spinach line SSB66-1090 F or spinach lineSSB66-1131 M, a sample of seed of said lines having been deposited underATCC Accession Number PTA-12018 and ATCC Accession Number PTA-12041,respectively.
 2. A seed comprising at least a first set of thechromosomes of spinach line SSB66-1090 F or spinach line SSB66-1131 M, asample of seed of said lines having been deposited under ATCC AccessionNumber PTA-12018 and ATCC Accession Number PTA-12041, respectively. 3.The plant of claim 1, which is inbred.
 4. The plant of claim 1, which ishybrid.
 5. The plant of claim 4, wherein the hybrid plant is spinachhybrid RX 06681616, a sample of seed of said hybrid RX 06681616 havingbeen deposited under ATCC Accession Number PTA-12059.
 6. The plant ofclaim 1, wherein the plant is a plant of line SSB66-1090 F or lineSSB66-1131 M.
 7. A plant part of the plant of claim
 1. 8. The plant partof claim 7, further defined as a fruit, a ovule, pollen, a leaf, or acell.
 9. A spinach plant having all the physiological and morphologicalcharacteristics of the spinach plant of claim
 5. 10. A spinach planthaving all the physiological and morphological characteristics of thespinach plant of claim
 6. 11. A tissue culture of regenerable cells ofthe plant of claim
 1. 12. The tissue culture according to claim 11,comprising cells or protoplasts from a plant part selected from thegroup consisting of embryos, meristems, cotyledons, pollen, leaves,anthers, roots, root tips, pistil, flower, seed and stalks.
 13. Aspinach plant regenerated from the tissue culture of claim 12, whereinsaid plant comprises all of the morphological and physiologicalcharacteristics of the spinach plant of claim 1, when grown under thesame environmental conditions.
 14. A method of vegetatively propagatingthe plant of claim 1 comprising the steps of: (a) collecting tissuecapable of being propagated from a plant according to claim 1; (b)cultivating said tissue to obtain proliferated shoots; and (c) rootingsaid proliferated shoots to obtain rooted plantlets.
 15. The method ofclaim 14, further comprising growing at least a first plant from saidrooted plantlets.
 16. A method of introducing a desired trait into aspinach line comprising: (a) crossing a plant of line SSB66-1090 F orSSB66-1131 M with a second spinach plant that comprises a desired traitto produce F₁ progeny, a sample of seed of said lines having beendeposited under ATCC Accession Number PTA-12018, and ATCC AccessionNumber PTA-12041, respectively; (b) selecting an F₁ progeny thatcomprises the desired trait; (c) backcrossing the selected F₁ progenywith a plant of line SSB66-1090 F or SSB66-1131 M to produce backcrossprogeny; (d) selecting backcross progeny comprising the desired traitand the physiological and morphological characteristics of spinach lineSSB66-1090 F or SSB66-1131 M; and (e) repeating steps (c) and (d) threeor more times to produce selected fourth or higher backcross progenythat comprise the desired trait.
 17. A spinach plant produced by themethod of claim 16, wherein the plant comprises the desired trait andotherwise comprises essentially all of the morphological andphysiological characteristics of the plant of line SSB66-1090 F or lineSSB66-1131 M , when grown under the same environmental conditions.
 18. Amethod of producing a plant comprising an added trait, the methodcomprising introducing a transgene conferring the trait into a plant ofhybrid RX 06681616, line SSB66-1090 F or line SSB66-1131 M, a sample ofseed of said hybrid and lines having been deposited under ATCC AccessionNumber PTA-12059, ATCC Accession Number PTA-12018, and ATCC AccessionNumber PTA-12041, respectively.
 19. A plant produced by the method ofclaim 18, wherein the plant has the transgene and otherwise hasessentially all of the morphological and physiological characteristicsof the plant of hybrid RX 06681616, line SSB66-1090 F or line SSB66-1131M, when grown under the same environmental conditions.
 20. The plant ofclaim 1, further comprising a transgene.
 21. The plant of claim 20,wherein the transgene confers a trait selected from the group consistingof male sterility, herbicide tolerance, insect resistance, pestresistance, disease resistance, modified fatty acid metabolism,environmental stress tolerance, modified carbohydrate metabolism andmodified protein metabolism.
 22. The plant of claim 1, furthercomprising a single locus conversion.
 23. The plant of claim 22, whereinthe single locus conversion confers a trait selected from the groupconsisting of male sterility, herbicide tolerance, insect resistance,pest resistance, disease resistance, modified fatty acid metabolism,environmental stress tolerance, modified carbohydrate metabolism andmodified protein metabolism.
 24. A method for producing a seed of aplant derived from at least one of hybrid RX 06681616, line SSB66-1090 For line SSB66-1131 M comprising the steps of: (a) crossing a spinachplant of hybrid RX 06681616, line SSB66-1090 F or line SSB66-1131 M withitself or a second spinach plant; a sample of seed of said hybrid andlines having been deposited under ATCC Accession Number PTA-12059, ATCCAccession Number PTA-12018, and ATCC Accession Number PTA-12041,respectively; and (b) allowing seed of a hybrid RX 06681616, lineSSB66-1090 F or line SSB66-1131 M-derived spinach plant to form.
 25. Themethod of claim 24, further comprising the steps of: (c) selfing a plantgrown from said hybrid RX 06681616, SSB66-1090 F or SSB66-1131 M-derivedspinach seed to yield additional hybrid RX 06681616, line SSB66-1090 For line SSB66-1131 M-derived spinach seed; (d) growing said additionalhybrid RX 06681616, line SSB66-1090 F or line SSB66-1131 M-derivedspinach seed of step (c) to yield additional hybrid RX 06681616, lineSSB66-1090 F or line SSB66-1131 M-derived spinach plants; and (e)repeating the crossing and growing steps of (c) and (d) to generate atleast a first further hybrid RX 06681616, line SSB66-1090 F or lineSSB66-1131 M-derived spinach plant.
 26. The method of claim 24, whereinthe second spinach plant is of an inbred spinach line.
 27. The method ofclaim 24, comprising crossing line SSB66-1090 F with line SSB66-1131 M,a sample of seed of said lines having been deposited under ATCCAccession Number PTA-12018, and ATCC Accession Number PTA-12041,respectively.
 28. The method of claim 25, further comprising: (f)crossing the further hybrid RX 06681616, SSB66-1090 F or SSB66-1131M-derived spinach plant with a second spinach plant to produce seed of ahybrid progeny plant.
 29. A hybrid seed produced by the method of claim27.
 30. A plant produced by growing the seed of claim
 27. 31. A plantpart of the plant of claim
 30. 32. The plant part of claim 31, furtherdefined as a fruit, a flower, a leaf, an ovule, pollen, or a cell.
 33. Amethod of producing a spinach seed comprising crossing the plant ofclaim 1 with itself or a second spinach plant and allowing seed to form.34. A method of producing a spinach leaf comprising: (a) obtaining aplant according to claim 1, wherein the plant has been cultivated tomaturity; and (b) collecting a spinach from the plant.